Forensic DNA extraction from decomposed human soft tissues: Optimization using ethanol treatment and surfactant-based...

Recovery of usable DNA from postmortem human remains is frequently limited by extensive degradation due to decomposition, which may compromise downstream genetic profiling. This study aimed to optimize DNA extraction from decomposed human soft tissues by evaluating a modified protocol adopting chilled ethanol treatment and/or surfactant-based lysis and chaotropic binding chemistry. A total of 225 soft tissue samples from eight organs (heart, liver, kidney, lung, brain, pectoralis muscle, iliacus muscle, and uterus) were obtained from 30 forensic autopsy cases representing across postmortem stages. Three extraction protocols were compared: a standard silica-column method, a modified protocol with chilled ethanol pretreatment, and a combined protocol with ethanol pretreatment and alternative buffers, such as SDS/Triton X-100 surfactant lysis buffer and a guanidine hydrochloride-based binding buffer. DNA yield and purity were assessed using spectrophotometry, while double-stranded DNA concentration and degradation index were evaluated using fluorometric quantification and quantitative PCR. STR profiling performance was examined by analyzing mismatch, drop-in, and drop-out rates as well as peak heights. Ethanol pretreatment significantly increased DNA yield across most tissue types and postmortem stages, while the combined protocol further improved DNA recovery and purity. Fluorometric and qPCR analyses confirmed higher double-stranded DNA concentrations under the modified protocols compared with the standard method. Differences in the degradation index were limited, indicating that the modified protocols produced balanced recovery of short and long DNA fragments. STR analysis demonstrated reduced mismatch, drop-in, and drop-out rates and higher peak heights across most tissue types and postmortem stages, with the benefits becoming more pronounced as decomposition progressed. These findings indicate that optimized extraction strategies can enhance STR profiling success from decomposed remains and broaden the range of postmortem specimens suitable for forensic genetic analysis from traditionally used hard tissues to a wider range of soft tissues.